植物二倍半萜生物合成途径与调控研究

The natural sesterterpenoids, a small subgroup of terpenoids derived from five isoprene units, are mostly isolated from marine organisms,but barely found in plants. Most sesterterpenoids feature unprecedented structural complexity and fascinating bioactivities. Their biosynthesis has not been well investigated, especially in plants. Our previous studies on two Lamiaceae plants, Leucosceptrum canum and Colquhounia coccinea var. mollis, led to the discovery of two series of unique sesterterpenoids, which are highly accumulated in the glandular trichomes and function as defense compounds. These sesterterpenoids share similarity in structural features due to their common 5/6/5 core structure, but are clearly distinct by their adverse stereochemistries and side chain modification. Those discoveries present us with a tremendous opportunity to study plant sesterterpeniods biosynthesis pathway and regulation. Within the NSFC supported project, investigation on the biosynthesis of GFPP (Geranylfarnesyl diphosphate), the precursor of sesterterpenoids, resulted in the clone and functional characterization of the GFPPS (GFPP synthase) in L. canum. Based on our previous researches, this project aims to further investigate the biosynthesis pathway and regulation of plant sesterterpeniods: to clone and indentify GFPPS and sesterterpeniod synthase, and then reveal the committed steps of plants sesterterpeniods biosynthesis; to study the expression of GFPPS and sesterterpeniod synthase and the content of sesterterpeniods in two plants under biotic and abiotic stresses, and then uncover the regulation of sesterterpenoids biosynthesis in transcription level; to investigate the function of GFPPS and sesterterpeniod synthase interacting proteins and the related signal transduction pathways, and then elucidate the regulation of sesterterpenoids biosynthesis in protein level. The ultimate aims of this project are expected to provide theoretic and scientific bases for the utilization of these sesterterpeniods.

天然二倍半萜结构复杂、活性广泛，主要分布于海洋生物中，植物中较为少见。二倍半萜生物合成研究很少，植物中未见报道。前期，本课题组发现唇形科植物米团花和火把花的腺毛能高度富集新奇的防御性二倍半萜，其核心骨架均为5/6/5三环体系，但立体化学及侧链修饰却截然不同；并在NSFC青年基金的资助下，对米团花二倍半萜生物合成前体GFPP的形成进行了研究，克隆、鉴定了GFPP合成酶基因。在此基础上，本项目拟深入开展植物二倍半萜生物合成途径与调控研究。以米团花和火把花的腺毛为材料，结合转录组测序等手段，克隆并鉴定二倍半萜生物合成的关键酶：GFPP合成酶(火把花)和二倍半萜合成酶基因，解析植物二倍半萜生物合成的主要途径；研究生物和非生物胁迫后关键酶基因的表达量和二倍半萜的含量，分析植物体内与关键酶相互作用蛋白的功能和信号转导通路，阐明二倍半萜生物合成的调控机制，为揭示植物二倍半萜的生物学功能提供科学依据。

本项目对两种富含二倍半萜化合物的唇形科植物米团花和火把花二倍半萜的生物合成进行了研究。从米团花的腺毛中克隆并功能鉴定了1个二倍半萜生物合成途径的关键酶——香叶基法尼基焦磷酸酯合成酶（GFDPS），能够特异性地催化二倍半萜C25直链前体GFDP的生成，并发现植物二倍半萜的生源途径是定位于质体中的MEP途径。此外发现茉莉酸甲酯能够诱导米团花中GFDPS的表达和二倍半萜的积累，同时提高植物的抗虫防御功能。进化分析发现植物GFDPS可能起源于正选择下GGDPS（二萜C20直链前体GGDP的合成酶）的复制和新功能化。从米团花中鉴定了1个二倍半萜合酶基因，其主要酶活产物为4个大环二倍半萜，包括2个新化合物。生物活性测试发现这些酶活产物具有一定的免疫抑制和肿瘤细胞毒活性。此外还鉴定了3个单萜合酶（β-罗勒烯合酶、柠檬烯合酶和α-蒎烯合酶）和4个倍半萜合酶（β-石竹烯合酶、雪松醇合酶、α-法尼烯合酶和selina-4,11-diene合酶）。从火把花中鉴定了1个GFDPS、9个GGDPS、1个单萜合酶（CcTPS2为linalool合酶）、3个多功能倍半萜合酶（β-eudesmol合酶、α-cedrene/γ-curcumene合酶、germacrene D合酶）、1个柯巴基焦磷酸酯合酶、2个角鲨烯合酶、以及1个多功能的单萜/倍半萜/二萜/二倍半萜合酶。另外本项目构建了萜类化合物合成生物学微生物体系。研究结果为深入研究二倍半萜在植物体内的生物合成途径与调控、生物功能及其应用奠定了基础，并为萜类化合物的合成生物学研究奠定了重要基础。目前发表带标注的SCI论文2篇，包括1篇 Plant Cell（5年平均影响因子为9.378）的封面文章，参加国内外学术会议交流11次，协助培养博士研究生3名和硕士研究生3名，项目负责人由助理研究员晋升为副研究员，并入选中科院“青年创新促进会”会员，中科院“西部青年学者”A类和云南省“万人计划”拔尖人才。